This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Biologically, the hydroxylation of methane is carried out by methane monoxygenases(MMO) found in methantrophic bacteria. The soluble form of MMO utilizes a diiron active site, which reacts with dioxygen to generate a -peroxo-Fe(III)Fe(III) species (MMO-P) and then an Fe(IV)Fe(IV) species (MMO-Q), which is responsible for oxidizing methane. The intermediates of the MMO catalytic cycle have been the subject of intense experimental and theoretical studies. However, MMO-Q and MMO-P have eluded structural characterization, and many questions about the nature of these intermediates remain. The experimental data for MMO-P have been used to argue for a cis-mu-1,2-peroxo bridging mode, while computational studies favor a mu-2:2-O2 core. For MMO-Q the 2.5 [unreadable] Fe-Fe distance from EXAFS favors a bis-mu-oxo Fe(IV)-Fe(IV) diamond core structure. However, no vibrational data exist to support any of the postulated core structures and the mechanism for conversion of MMO-P to MMO-Q is unknown. The proposed research is aimed at elucidating the electronic structure of these key intermediates in methane hydroxylation by utilizing new spectroscopic methods. K-Beta x-ray emission spectroscopy is proposed as a selective probe of the Fe2O2 upper valence region, which should provide a sensitive measure of the oxygen bond strength and the binding mode. These data will be complemented by conventional XAS data and correlated to DFT calculations of the spectra. Our initial studies will focus on well characterized model complexes and will then be extended to the enzymatic intermediates. The results of these studies should provide fundamental insights into the biological process of methane hydroxylation.